In the last 15 years, the incidence of Type 2 diabetes associated with cardiovascular disease has more than doubled. The underlying pathology includes both endothelial and vascular smooth muscle (VSM) dysfunction which we have observed in two different mouse models of diabetes: leptin receptor deficient (db/db) and streptozotocin-induced. Compared to non-diabetic mice, aorta from both diabetic mouse models relax less to endothelial NO-mediated vasodilators and contract more to serotonin and other vasoconstrictors, in part, mediated by increased activation of the rho/rho kinase pathway. Although studies have demonstrated increased contractile function in diabetes, the cellular mechanisms underlying this abnormality are unknown. The long-term goal of my research is to understand the cellular mechanisms that contribute to the dysfunction of VSM in diabetes. Vasoconstrictor serotonergic receptors and rhoA have been found associated with caveolin-1 in specialized plasma membrane microdomains of VSM. Studies in our laboratory demonstrate that contractions of aorta isolated from caveolin-1 (cav-1) gene deficient mice are markedly increased to serotonin which are restored to normal by inhibitors of rho kinase. Rho-dependent contractions are similarly increased in diabetes. Our overall hypothesis is that serotonin receptors and their downstream signaling molecules are localized within caveolae in an inactive state. This sequestering of receptors and rhoA in caveolae is reduced in diabetes contributing to greater activation and contraction of VSM. In this proposal we will assess the impact of diabetes on the expression of rhoA, rho kinase and serotonin receptors in caveolar microdomains. We will also determine if the subtype of serotonin receptors changes in VSM in diabetics (streptozotocin- induced or leptin receptor deficient). Finally, we will determine whether a change in the expression of specific receptor subtypes or their downstream signaling components within caveolae contributes to the enhanced vasoconstriction in diabetes. To test this hypothesis, we will address the following specific aims: SPECIFIC AIM 1: To establish if receptor-mediated activation of the rho/rho kinase pathway occurs in caveolar microdomains in vascular smooth muscle. Our working hypothesis is that receptors that activate the rho/rho kinase pathway are sequestered in caveolae. We will assess expression and localization of specific serotonin receptors, rhoA and rho kinase in caveolar microdomains in aorta from wild type and caveolin-1 deficient mice under basal and stimulated conditions. SPECIFIC AIM 2: To determine whether VSM caveolar complexes change in diabetes to increase receptor signaling through the rho/rho kinase pathway. Our working hypothesis is that localization of serotonin receptors and/or rhoA in caveolar microdomains suppresses their activation which is reduced in diabetes. We will compare expression of serotonin receptors, rhoA and rho kinase in association with cav-1 in lipid rafts in non-diabetic and diabetic wild type or cav-1 deficient aorta under basal and stimulated conditions. SPECIFIC AIM 3: To determine whether the vascular dysfunction in diabetes is related to a change in caveolin-1 compartmentalization of serotonin receptors or rhoA. Our working hypothesis is that deletion of caveolin-1 will augment receptor mediated rhoA activation and this is increased in diabetic arteries. We will compare the effects of inhibition (siRNA) or overexpression of caveolin-1 using viral transfection on vascular responses to specific serotonin receptor agonists or direct activation of rhoA in non-diabetic and diabetic mice. PUBLIC HEALTH RELEVANCE: The incidence of diabetes that is associated with cardiovascular disease has more than doubled in the past 15 years. The objective of this proposal is to determine if the smooth muscle dysfunction underlying the pathology of diabetic vascular disease is related to a change in the localization of signaling molecules that determine the magnitude of smooth muscle contraction in diabetic blood vessels.